1. Field of the Invention
This invention relates to theobromine synthase polypeptide and the gene encoding said enzyme.
2. Prior Art
Coffee is a drink consumed all over the world with favorite and its utility is markedly large. On the other hand, it is known that excessive ingestion of caffeine, which is contained in coffee, causes harmful effects. Caffeine is one of xanthine derivatives and theophylline and theobromine are also the members of the xanthine derivatives. These xanthine derivatives are known to inhibit phosphodiesterase, thereby the amount of cAMP is increased. As the result, xanthine derivatives exhibit excitatory effect on the central nerves system and enhance function of the circulatory system. When they are ingested at a suitable amount, such effects of xanthine derivatives are useful for spiritual elevation. However, when the amount of digestion is excessive, they would cause harmful effects as mentioned above. Therefore, there has been a strong demand on production of a caffeine-less coffee all over the world.
To obtain caffeine-less coffee, attempts to obtain a gene involved in biosynthesis of xanthine derivatives have been performed, in the purpose to achieve artificial control of biosynthesis of caffeine. In FIG. 1 (cited from Advances in Botanical Research, Vol. 30, Academic Press (1999) p149), the pathway working for caffeine biosynthesis in coffee plants is shown. In FIG. 1, the arrow with solid line indicates the main pathway of caffeine synthesis and the arrow with dotted line indicates the minor pathway of caffeine synthesis, respectively. As shown in the second line of FIG. 1, the pathway operating for biosynthesis of caffeine from xanthosine via 7-methylxanthine and theobromine has been known, which is the main pathway for biosynthesis of caffeine biosynthesis in coffee plants. The latter half of the main biosynthesis pathway of caffeine is composed of three steps of N-methylation reactions. These N-methylation reactions have been known to be dependent on S-adenosylmethoinine. There also exists a pathway (third line in FIG. 1) in which caffeine is biosynthesized from 7-methylxanthine via para-xanthine, but it is known that contribution of this pathway is not significant. With regard to the first methylation reaction to synthesize 7-methylxanthine, a gene encoding an enzyme responsible for said reaction has been obtained and it has been already reported (International Laid-Open Publication No. WO 97/35960). However, genes involved in the second step methylation reaction and the third step methylation reaction have not been known yet. For effective and accurate manipulation of caffeine biosynthesis, more knowledge on genes that encode enzymes involved in caffeine biosynthesis should be obtained.
The first aspect of this invention is a polypeptide consisting of an amino acid sequence defined by amino acid numbers from 1 to 378 shown in SEQ ID NO: 1 in a Sequence List. A polypeptide consisting of an amino acid sequence exhibiting at least 90% of homology with SEQ ID NO: 1 is also within the scope of this invention, so far as the polypeptide has the activity to biosynthesize theobromine using 7-methylxanthine as the substrate. Such sequence may be obtained by making deletions, insertions, substitutions or any combinations thereof in the amino acid sequence of SEQ ID NO: 1.
The second aspect of this invention is a gene consisting of a base sequence defined by base numbers from 1 to 1298 shown in SEQ ID NO: 2 in a Sequence List. A gene that hybridizes with SEQ ID NO: 2 under a stringent confdition and a gene consisting of a base sequence exhibiting at least 90% of homology with SEQ ID NO: 2 is also within the scope of this invention, so far as the gene encodes a polypeptide having the activity to biosynthesize theobromine using 7-methylxanthine as the substrate. Such sequence may be obtained by making deletions, insertions, substitutions or any combinations thereof in the base sequence of SEQ ID NO: 2.
The third aspect of this invention is a transformed plant wherein expression of said gene is inhibited in the plant to decrease biosynthesis of theobromine and a seed obtained from the transformed plant. Preferably, the plant to be transformed is selected from the group consisting of Coffea arabica, Coffea canephora, Coffea liberica and Coffea dewevrei. 
The fourth aspect of this invention is a transformed plant wherein said gene is introduced in the plant to increase biosynthesis of theobromine and a seed obtained from the transformed plant. Preferably, the plant to be transformed is selected from the group consisting of Coffea arabica, Coffea canephora, Coffea liberica and Coffea dewevrei.